Phytic acid takes up about 50-70% of phosphate contained in animal forages. However, monogastric animals such as fowls and pigs lack digestive enzymes for separating inorganic phosphorus from the phytic acid molecule, so that a coefficient of utilization of phosphorus is very low. Phosphate of phytic acid is not absorbed, passes through the digestive tract and is excreted. This leads to an increased ecological phosphorus burden to land and water. In addition, since phytate chelates several essential minerals and prevents or inhibits their absorption in the digestive tract, phytic acid decreases the nutritional value of food and animal feeds.
Obviously, phosphorous (P) is an essential element for growth, so that inorganic phosphorus (e.g., dicalcium phosphate, defluorinated phosphate) or animal products (e.g., meat and bone meal, fish meal) are added to meet the animals' nutritional requirements for phosphorus, and it is very expensive.
Phytases, a specific group of monoester phosphatases, are required to initiate the release of phosphate (“P”) from phytic acid (myo-inositol hexophosphate), the major storage form of P in cereal foods or feeds (E Graf et al. 1987). Phytase is widely distributed in plants, animals and microorganisms. Based on the characteristics of phytases from different organisms, microbial phytase are getting more and more attention. And, microbial phytase, as a feed additive, has been found to improve the bioavailability of phytate phosphorous in typical monogastric diets (Cromwell, et al, 1993). The result is a decreased need to add inorganic phosphorous to animal feeds, as well as lower phosphorous levels in the excreted manure (Kornegay, et al, 1996). With the development of gene engineering, on the one hand, more and more microbial phytases have been isolated and/or purified. For example, “Purification and Characterization of a Phytase from Klebsiella terrigena,” (Greiner et al. 1997), “Purification and Properties of a Thermostable Phytase from Bacillus sp. DS11,” (Kim et al. 1998), “Isolation and characterization of a phytase with improved properties from Citrobacter braakii” (Kim et al 2003), and “Gene cloning, expression and characterization of novel phytase from Obesumbacterium proteus” (Zinin et al 2004); on the other hand, improved properties of the conventional phytase have been achieved by Site-directed mutagenesis or gene shuffling. For example, Improving thermostability of Aspergillus niger phytase by elongation mutation (chen et al, 2005), or Site-directed mutagenesis of Escherichia coil phytase (Lei X G, 2005). So manufacture costs of microbial phytase was reduced largely. Because of these advantages, some of the known phytases have gained widespread acceptance in the feed industry.
However, problems still exist in these known phytases. Because these phytases do not react ideally as an additive in feed, most phytases have completely lost their activity during feed pelleting process and are unable to degrade phytic acid in stomach or intestines. The reasons for this vary from enzyme to enzyme. Typical concerns relate to poor stability and low activity of the enzyme in the environment of the desired application. For example, the temperature encountered in the processing of feedstuffs, the pH and the proteases in the digestive tracts of animals, and the degradation during storage.
It is, thus, generally desirable to discover and develop novel enzymes having good stability and phytase activity for use in connection with animal feed, and to apply advancements in fermentation technology to the production of such enzymes in order to make them commercially viable.
On the other hand, conventional methods to obtain a new phytase gene are mainly based on screen form genomic library or direct separation of proteins. However, the conventional methods were very laborious, difficult and expensive. And it is well known that phytases are of relatively low homology among different species, which present a challenge for traditional approach to isolate a phytase gene.